Lignin content has been an obstacle to efficient transformation of lignocellulosic biomass to bioethanol, feeds, organic precursors and other useful products. Enzymatic conversion of lignocellulosic polysaccharides to fermentable sugars typically requires high enzyme loadings to overcome comparatively low efficiencies of enzymatic catalysis. The comparatively inefficient hydrolysis of cellulosic substrates in pre-treated lignocellulosic biomass is partly due to obstruction by lignin of enzyme access to catalytically productive cellulose binding. The precise nature of this obstruction is not completely understood but apparently arises from the complex and hydrophobic nature of lignin and from its intimate distribution between cellulose fibers.
Part of the deleterious effect of lignin on cellulose hydrolysis in pre-treated lignocellulosic biomass is due to “non-productive” binding of cellulase enzymes to lignin-rich residual material that lacks polysaccharides. See ref. 1. Another aspect is that lignin provides a physical-chemical barrier to catalytically productive enzyme binding with cellulose substrates, further limiting hydrolysis rates. See ref. 2. Lignin-containing pre-treated biomass exhibited roughly equivalent cellulase binding in mg/g substrate compared with de-lignified biomass. However, lignin-containing substrates exhibited much lower initial rates of hydrolysis—lower by a factor of at least three from hydrolysis rates achieved with de-lignified substrates. This reduction in hydrolysis rates could not be explained solely by “non-productive” enzyme binding to lignin. Some additional “lignin barrier” to enzyme access was clearly implicated. The precise nature of this “lignin barrier” remains unclear. Catalytically productive binding of cellulase enzymes to cellulosic substrates has been studied in detail and is known to involve physical-chemical subtleties. Lu et al (ref 2) suggest that “re-swelling” properties of cellulose fibers in water have some influence on cellulose structure, enzyme adsorption capacity and enzymatic hydrolysis by cellulases. A functional effect of cellulose fiber “swelling properties” is suggested by the much higher cellulase binding exhibited by pre-treated lignocellosic substrates (both lignin containing and de-lignified) compared with pure microcrystalline cellulose (AVICEL™), which is produced by processes involving drying and bleaching. Further, amorphous regions of cellulose fibers provide greater opportunities for catalytically productive cellulase binding compared with crystalline regions of cellulose fibers. See e.g ref. 3 and 4.
One approach to reducing deleterious effects of lignin has been de-lignification of pre-treated lignocellulosic biomass. See e.g ref. 5 and 6. Another approach to the lignin problem which has been explored on an experimental scale has been introduction of additives to hydrolysis mixtures, including surfactants, proteins and other lignin-binding polymers. In particular, polyethylene glycol (PEG) of varying molecular weights has shown promise. Most of the surfactants tested previously were similar to PEG in that they included ethylene oxide core structures, such as TWEEN™. See ref. 7, 8, 9, 10 and 11.
Efforts to “scale up” the use of “lignin binding” additives from an experimental scale to production scale have focused on the study of molecular mechanisms of the surfactant/PEG effect on cellulose hydrolysis rates. See ref. 12 and ref. 13. Börjesson et al confirmed the conclusions of previous studies by Sewalt et al (1997) (ref. 7) and by Erikkson et al (2002) (ref. 10) that the surfactant/PEG effect primarily involves reversal of catalytically unproductive enzyme binding to lignin. Börjesson et al note that this conclusion is consistent with observations of effects comparable to the PEG effect achieved by addition of bovine serum albumin, which is widely used to suppress non-specific protein binding in various experimental contexts. See ref. 14.
With the express aim of identifying optimal conditions for PEG/surfactant addition, Börjesson et al. studied the surfactant/PEG effect in close detail, including binding isotherms of PEG 4000 with steam pre-treated spruce. Börjesson et al. concluded that optimal PEG conditions could be achieved by addition of about 0.05 g PEG (g DM)−1. These optimal PEG conditions are in good agreement with results reported by other researchers. Ref. 15 reports that optimal hydrolysis conditions could be achieved using steam and acid pre-treated corn stover by addition of about 0.1 g (g DM)−1 of non-ionic, ethylene oxide polymer surfactants selected from the group consisting of SOFTANOL™ 50, SOFTANOL™ 90, SOFTANOL™ 120, SOFTANOL™ 200, LUTSENOL™ AT50, LUTSENOL™ AT80, TERGITOL™ NP9, NOVELL II™ TDA 6.6, NOVELL II™ TDA 8.5, BRIJ™ 35, BRIJ™ 56, BRIJ™ 97, BRIJ™ 98, and PLURONIC™ F68. Ref. 16 reports that optimal hydrolysis conditions could be achieved using wheat straw pre-treated by water and by sulfuric acid by addition of about 0.05 g (g DM)−1 of either BEROL™ 08, PEG 6000, TWEEN™ 80 or BSA. Ref. 10 reports hydrolysis conditions using steam treated spruce with about 0.05 g (g DM)−1 of non-ionic surfactants including TWEEN™ 20, TWEEN™ 80, TRITON™ X-100, TRITON™ X-114, AGRIMUL™ NRE 1205, and hydrophobically modified ethylene oxide co-polymer. Ref. 17 reports hydrolysis using steam exploded wheat straw with about 0.05 g (g DM)−1 of TWEEN™ 20.
Here we report the surprising discovery that, when hydrolysis of pre-treated lignocellulosic biomass is conducted at high dry matter content, above 20%, optimal PEG/surfactant conditions can be achieved at lower levels than those known in the prior art, or about 0.025 g (g DM)−1. The lowered optimal PEG/surfactant conditions enables cost-saving reductions in consumption of additives in bioethanol production at high dry matter content. Even sub-optimal PEG/surfactant conditions, as low as 0.01 g (g DM)−1, provide satisfactory results at high dry matter. An immediate implication of this result is that the molecular mechanism of the PEG/surfactant effect is likely not a simple matter of competitive inhibition of catalytically non-productive lignin binding, but rather involves potentially intricate details of inter-fiber surface chemistry.
We further report that this effect is most pronounced at comparatively low cellulose loadings, <7 FPU (g DM)−1. Thus, hydrolysis yields can be improved and enzyme requirements reduced at high dry matter by adding surprisingly small quantities of PEG or surfactant.
Hydrolysis mixtures prepared using high dry matter content, in the presence of PEG/surfactant, can be readily used in simultaneous saccharification and fermentation (SSF) and other processes involving fermentation in the presence of lignin.